The present invention is generally in the area of aqueous two phase systems for use in partitioning components in an aqueous solution.
The study of biological systems requires efficient means of separation which are mild enough such that biological activity is retained throughout the process. Aqueous two-phase systems, which are both mild and efficient, have been in use in laboratories for about thirty years. Per-Ake-Albertsson, Partition of Cell Particles and Macromolecules, 3rd Edn., John Wiley & Sons, New York, 1986. Aqueous two phase systems form when two water soluble polymers are dissolved in water. The aqueous two phase systems contain mainly water, with the first polymer predominating in one phase and the second polymer predominating in the other phase. Due to their high water content, both equilibrium phases provide a suitable environment for biological macromolecules. When a mixture of proteins is added to an aqueous two phase polymer system, each type of protein partitions uniquely between the two phases.
There has been an increase in interest in this type of system, both industrially and in research applications, during the past few years. Walter, H. and Johansson, G., Eds., Methods in Enzymology, Vol. 228 (1994). The system can be used to separate a desired product from a cell culture. For example, an enzyme can be partitioned into one easily-removable phase while cell debris and unwanted molecules remain in the other phase. Alternatively, an enzyme can be confined to one phase and supplied with a substrate, the products of the fermentation of that substrate being extracted into the other phase. This process is a form of extractive bioconversion.
Applications of this methodology in the biotechnology industry are described in a review by Hustedt et al., and economic considerations have been discussed by Kroner et al. Hustedt et al., "Applications of Phase Partitioning in Biotechnology", in "Partitioning in Aqueous Two-Phase Systems: Theory, Methods, Uses and Applications to Biotechnology", Academic Press, New York, 1985; and Kroner et al., Process Biochem., 19:170-179 (1984). Kroner et al. concluded that aqueous two phase conversion was economically the best method for production of enzymes providing that the cost of chemicals for creation of the two phases could be contained by recycling or reduction in the amount required by modification of the phase systems.
Aqueous two phase systems formed from poly(ethylene glycol) (PEG) and a salt, usually potassium phosphate or ammonium sulfate, have long been used for purification of enzymes in the laboratory. This system is economically viable on an industrial scale due to the low cost of the chemicals required to make up the phase system. Tjerneld, "Aqueous two phase partitioning on an industrial scale", in Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications, J. Milton Harris, Ed., Plenum Press, New York, 1992. However, there are certain disadvantages with the PEG-salt system. The high concentrations of salt present can be tolerated by many proteins but not by a cell or organelle. Per-Ake-Albertsson, Partition of Cell Particles and Macromolecules, 3rd Edn., John Wiley & Sons, New York, 1986. To encompass such sensitive separations, a much milder system is required in which salt is replaced by another polymer. High concentrations of salt may also interfere with affinity partitioning in which a biospecific ligand is bound to the PEG-rich phase. Johansson, G., Methods Enzym., 228:64-74 (1994); and Johansson, G., "Affinity Partitioning in PEG-containing Two phase Systems", in Poly (Ethylene Glycol) Chemistry: Biotechnical and Biomedical applications, J. Milton Harris, Ed., Plenum Press, New York, 1992. On an economic industrial scale, high concentration of phosphate and sulfate in effluent streams pose an economic and environmental problem. Kroner et al., Process Biochem., 19:170-179 (1984); and Tjerneld, "Aqueous two phase partitioning on an industrial scale", in Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications, J. Milton Harris, Ed., Plenum Press, New York, 1992.
Aqueous two phase systems using two polymers offer a number of advantages over PEG-salt systems in the laboratory situation. Mixtures of unlike polymers in aqueous solution will generally separate into two phases with water contents ranging from 85-99%. Such a system is very mild, and the polymers appear to stabilize particle structure and biological activity. Mattiasson, B., Methods Enzym., 137:657-667 (1988). Additionally there is an extremely low interfacial tension which does not damage delicate particles. Reproducible partitioning of macromolecules, cells and cell particles can be obtained under mild conditions. Per-Ake-Albertsson, Partition of Cell Particles and Macromolecules, 3rd Edn., John Wiley & Sons, New York, 1986.
The most commonly used two-polymer phase systems are of the PEG/dextran ("PEG-dx") type, using a synthetic hydrocarbon ether polymer and a sugar polymer which are mutually incompatible. Although alternatives to PEG are available, it remains the most widely used synthetic polymer for laboratory and commercial applications. Bamberger et al., "Preparation of Phase Systems and Measurements of their Physicochemical Properties" in Partitioning in Aqueous Two Phase Systems: Theory, Methods, Uses and Applications to Biotechnology, Academic Press, New York, 1985.
Dextran is available in a range of molecular weight fractions, however economic considerations preclude the use of fractionated dextrans on a large scale. Tjerneld and Johannsson, Bioseparation, 1:255-263 (1990). Crude dextran has been used for large-scale processes including semi-continuous enzymic hydrolysis of a model cellulose substrate and the enzymic hydrolysis of starch. Kroner et al., Biotechnol. Bioeng., 24:1015-1045 (1982); Tjerneld et al., Biotechnol. Bioeng., 27:1036-1043 (1985); Tjerneld et al., Biotechnol., Bioeng., 27:1044-1050 (1985); and Larsson et al., Biotechnol. Bioeng., 33:758-766 (1989). In these systems, the enzymes are retained in the lower dextran-rich phase, while the product is removed in the upper PEG-rich phase, which may be filtered and recycled. The high molecular weight fractions in crude dextran adversely affect the partitioning of the enzyme to the bottom phase so that some is lost in the top phase. This may be partially offset by the use of higher molecular weight PEG and the fact that lower concentrations are required for separation. However, the systems are considerably more viscous than those using fractionated dextrans. Moreover, the use of crude dextran only results in a four-fold reduction in cost over the use of fractionated dextrans. Kroner et al., Process Biochem., 19:170-179 (1984). Hydroxypropyl starch (HPS) derivatives have been evaluated as possible replacements for fractionated dextrans. Ling et al., Carbohydr. Polymers, 11:43-54 (1989); and Sturesson et al., Appl. Biochem. Biotech., 26:281-295 (1990).
Pullulan is a microbial polysaccharide which has been used in the form of a PEG-pullulan system for semi-continuous production of cellulases as well as batch separations of enzymes. Nguyen et al., Appl. Microbiol. Biotechnol., 27:341-346 (1988). Pullulan, however, can cause viscosity difficulties at higher concentrations. Maltodextrins from corn starch (molecular weight average 1200, 1800, 3600) have been proposed as a low cost alternative to fractionated dextrans. Szlag et al., ACS Symposium Series 419:71-86 (1990); and Szlag and Giuliano, Biotechnol. Techniques, 2:277-282 (1988). Because of the lower molecular weight of the maltodextrins, higher concentrations are required to achieve phase separation. The maltodextrins also are susceptible to starch degrading enzymes, which could be a significant consideration in industrial scale bioconversion in which the sugar polymer would be exposed to the enzyme for long periods.
There is a need for the development of improved aqueous two phase systems for the partitioning of biological materials. It is therefore an object of the invention to provide methods for separating biological materials from cells and other debris using aqueous two phase systems. It is another object of the invention to provide aqueous two phase extraction systems containing enzymes for use in bioconversion applications. It is a further object of the invention to provide aqueous two phase systems which can be used to partition biological materials such as cells and macromolecules without degrading or modifying the materials, and without affecting biological activity of the materials.